Unveiling the regulatory role of GRP7 in ABA signal-mediated mRNA translation efficiency regulation

Zhang, Jing; Shao, Wenna; Xu, Yongxin; Tian, Fa’an; Chen, Jinchao; Wang, Dongzhi; Lin, Xuelei; He, Chongsheng; Yang, Xiaofei; Staiger, Dorothee; Ding, Yiliang; Yu, Xiang; Xiao, Jun

doi:10.1038/s41467-025-59329-6

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Published: 26 April 2025

Unveiling the regulatory role of GRP7 in ABA signal-mediated mRNA translation efficiency regulation

Nature Communications volume 16, Article number: 3947 (2025) Cite this article

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Abstract

Abscisic acid (ABA) is a crucial phytohormone involved in plant growth and stress responses. While the transcriptional regulation triggered by ABA is well-documented, its effects on translational regulation have been less studied. Through Ribo-seq and RNA-seq analyses, we find that ABA treatment not only influences gene expression at the mRNA level but also significantly impacts mRNA translation efficiency (TE) in Arabidopsis thaliana. ABA inhibits global mRNA translation via its core signaling pathway, which includes ABA receptors, protein phosphatase 2Cs (PP2Cs), and SNF1-related protein kinase 2 s (SnRK2s). Upon ABA treatment, Glycine-rich RNA-binding proteins 7 and 8 (GRP7&8) protein levels decrease due to both reduced mRNA level and decreased TE, which diminishes their association with polysomes and leads to a global decline in mRNA TE. The absence of GRP7&8 results in a global impairment of ABA-regulated translational changes, linking ABA signaling to GRP7-dependent modulation of mRNA translation. The regulation of GRP7 on TE relies significantly on its direct binding to target mRNAs. Moreover, mRNA translation efficiency under drought stress is partially dependent on the ABA-GRP7&8 pathways. Collectively, our study reveals GRP7’s role downstream of SnRK2s in mediating translation regulation in ABA signaling, offering a model for ABA-triggered multi-route regulation of environmental adaptation.

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Introduction

Abscisic acid (ABA) is a crucial phytohormone that regulates plant development and responses to environmental stresses^1,2,3. The levels of ABA in plants are dynamically controlled through tightly regulated biosynthesis and metabolism^3,4. ABA biosynthesis involves multiple enzymatic steps, with the cleavage of carotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED), abscisic acid deficient 2 (ABA2), and abscisic aldehyde oxidase (AAO3)^3,4. Under abiotic stress conditions, such as drought and high salinity, specific signaling pathways are activated, leading to increased ABA accumulation^5,6.

The ABA signaling pathway is initiated by binding of ABA to its receptors, which include PYRABACTIN RESISTANCE1 (PYR1), PYRABACTIN-LIKE (PYL), and REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)^7,8. These receptors interact with PROTEIN PHOSPHATASE 2Cs (PP2Cs), forming a complex that inhibits the phosphatase activity of PP2Cs, thereby activating SNF1-RELATED PROTEIN KINASE 2s (SnRK2s)⁹. The activated SnRK2s then stimulate ABRE-binding protein/ABRE-binding factors (AREB/ABF), leading to enhanced transcription of ABA-responsive genes^1,10. In addition to facilitating downstream transcriptional regulation, research has indicated that ABA also suppresses global mRNA translation¹¹. Notably, the SUPPRESSOR OF THE ABAR OVEREXPRESSOR 1 (SOAR1) protein, a member of the pentatricopeptide repeat (PPR) family, inhibits the translation of ABA INSENSITIVE 5 (ABI5) by binding to its mRNA, thereby negatively regulating ABA signaling¹². Further research is essential to unravel the complex interplay between ABA signaling and translational regulation, enhancing the understanding of how ABA governs plant responses to environmental cues.

Translation is a dynamic process that involves interactions among mRNAs, transfer RNAs, and the ribosomal machinery, regulated by both cis- and trans-acting factors while integrating various internal and external signals^13,14. The ribosome serves as a complex molecular machine essential for catalyzing protein synthesis with speed and accuracy¹⁵. In plants, the cytoplasmic ribosome consists of two subunits: the 40S subunit, which decodes the mRNA, and the 60S subunit, responsible for the peptidyl transferase reaction¹⁶. The 40S subunit contains 18S rRNA and 33 ribosomal proteins, while the 60S subunit includes 5S, 5.8S, and 28S rRNAs, along with 47 ribosomal proteins^17,18.

Highly translated mRNAs are typically associated with more ribosomes (polysomes) than poorly translated mRNAs¹⁹. Polysome profiling is a common technique for analyzing the translational status of genes, where ribosomal occupancy (the percentage of transcripts associated with ribosomes) and ribosomal density (the number of ribosomes per mRNA) reflect translational efficiency²⁰. However, this method has limitations in resolution and accuracy²¹. To better understand translational regulation, it is crucial to measure the dynamic positional information and abundance of translating ribosomes on individual transcripts²². Ribosome profiling, which involves deep sequencing of the ribosome-protected mRNA fragments, was developed to map the positions of translating ribosomes on transcripts²¹. This technique has been instrumental in elucidating the molecular basis of translational regulation in photomorphogenesis²². Subsequent improvements in footprint precision and data quality by employing RNase T1 have enhanced its applicability^23,24, providing a rich foundation for studying translation regulation in detail²⁵.

Translational control plays a vital role in plant development^26,27 and stress responses^28,29. In rice, the RNA-binding protein MAO HUZI9 (MHZ9), which contains a glycine-tyrosine-phenylalanine (GYF) ___domain, responds to ethylene signals by binding to the 3’ untranslated region (UTR) of EIN3 BINDING F-BOX (EBF) mRNA. This interaction leads to translational inhibition and significantly contributes to downstream ethylene signaling pathways²⁸.

RNA-binding proteins (RBPs) are crucial for post-transcriptional regulation of RNAs, influencing various aspects of RNA metabolism, including splicing, stability, localization, and translation³⁰. They interact dynamically with RNA partners, participating in nearly all stages of RNA processing and function. The roles of many RBPs in plant development and stress responses have been documented³¹. Among these, glycine-rich RNA-binding proteins (GRPs) are particularly abundant and linked to abiotic stress responses in plants³². The GRP family consists of eight members, whose mRNA level can be induced by abiotic stresses. Under drought stress, the expression of GRP2, GRP4, and GRP7 decreases, while GRP3, GRP4, GRP6, and GRP7 show reduced expression under high salinity³³. GRP7, a well-studied member, shares high sequence similarity with GRP8 and exhibits functional redundancy with it³⁴. The grp7-1 8i mutant, which has an RNAi construct against AtGRP8 and expresses GRP8 at levels comparable to Col-0 plants, displays hypersensitivity to abscisic acid (ABA), and GRP7 protein levels significantly decline upon ABA treatment³⁵.

GRP7 features an N-terminal RNA recognition motif and a C-terminal glycine-rich ___domain that enables nucleocytoplasmic shuttling. It facilitates mRNA export from the nucleus to the cytoplasm during cold stress³⁶ and regulates flowering time by influencing RNA processing³⁷. GRP7 also affects RNA stability, impacting the circadian oscillation of the target mRNA LHCB1.1³⁸. Mutations in GRP7 have been shown to alter the content of both small RNAs (sRNA) and long non-coding RNAs (lncRNA) in apoplastic wash fluid (AWF), suggesting its involvement in the secretion and/or stabilization of exRNAs³⁹. Notably, GRP7 participates in innate immunity by translation regulation, interacting with eukaryotic translation initiation factors (eIF4A1, eIF4A2, eIF4E), and ribosomal protein uS11z⁴⁰. At elevated temperatures, GRP7 forms condensates in the cytoplasm through liquid–liquid phase separation, inhibiting the translation of GRP7 and HSP70-1⁴¹. However, the extent of GRP7’s role in global mRNA translation efficiency remains unclear.

In this study, we observed that ABA treatment not only induced significant mRNA level changes but also caused a multitude of alterations in translation efficiency in Arabidopsis thaliana, as detected by Ribo-seq and RNA-seq analyses. Furthermore, ABA treatment leads to a reduction in the protein levels of GRP7&8 through its core signaling factors. GRP7&8 can associate with ribosomes to influence mRNA translation efficiency and plays a crucial role in ABA signal-mediated translational regulation by directly binding to its target mRNAs. Additionally, the regulation of mRNA translation efficiency under drought stress is partially dependent on the ABA-GRP7&8 pathway.

Results

Differential regulation of gene expression and translation efficiency in response to ABA treatment

Plant exhibits rapid adjustments in gene expression at both transcriptional and translational levels in response to environmental stress, with the hormone ABA playing a pivotal role^42,43. By conducting RNA-seq and Ribo-seq analyses on three-day-old Col-0 seedling subjected to 5 μM ABA or mock treatment for 4 h, we explored the genes with differential mRNA and translation levels (Fig. 1a). As expected, strong correlation existed between steady-status RNA levels and translation levels within the same sample (Supplementary Fig. 1a). TE is calculated using normalized Ribo-seq reads divided by RNA-seq reads (see "Methods" for details).

**Fig. 1: ABA globally modulates translation efficiency.**

Under ABA treatment, we identified 5248 differentially expressed genes (DEGs) and 1548 genes exhibiting differential translation efficiency (DTE) (Fig. 1b and Supplementary Data 1, 2). Among these, 719 genes showed ABA-induced changes at both the mRNA and translational levels (Overlap), while 4529 genes were altered only at the mRNA level (DEG_only) and 829 genes only at the translational level (DTE_only) (Fig. 1c). Genes involved in the response to abscisic acid, cytokinin, cold, and mRNA binding exhibited changes at both levels (Fig. 1d). In contrast, genes related to ribosome biogenesis were primarily regulated at the mRNA level, whereas those involved in photosynthesis and chlorophyll biosynthesis were predominantly affected at the translational level (Fig. 1d). Notably, genes that were upregulated in both mRNA and translation efficiency were associated with responses to abiotic stresses (Supplementary Fig. 1b). Conversely, genes with downregulated translation efficiency were enriched in photosynthesis and chlorophyll biosynthetic process. Specifically, genes related to photosynthesis and chloroplast biogenesis, such as Chlorophyll A/B binding proteins 2 and 3 (CAB2, At1g29920; CAB3, At1g29910)^38,44 and Chloroplast Protein-Enhancing Stress Tolerance (CEST, At5g44650)⁴⁵, underscore the critical role of translational regulation in the ABA response (Fig. 1e). In contrast, PROTEIN PHOSPHATASE 2A SUBUNIT A3 (PP2A, At1g13320) served as a negative control, showing no changes in translation efficiency or RNA levels in response to ABA (Fig. 1e).

To further evaluate the translation efficiency of individual genes exhibiting differential TE under ABA treatment from Ribo-seq data, we measured mRNA levels in entire fractions of polysome profiling using RT-qPCR. The results were expressed as a percentage of the total RNA in each fraction. To quantify TE, we calculated the proportion of polysome-bound RNA (fractions 7–11) relative to the total RNA. This analysis confirmed that the TE of the photosynthesis genes CAB2, CAB3, and CEST decreased upon ABA treatment in Col-0, while the negative control gene PP2A showed no significant changes (Fig. 1f).

In summary, ABA treatment in Col-0 seedlings caused significant changes in gene expression, revealing the role of translational regulation alongside transcriptional regulation.

ABA signaling pathway components regulate global mRNA translation efficiency

We examined the impact of ABA signaling pathway factors on mRNA translation efficiency by focusing on the ABA receptor mutant pyr1pyl1/2/4^46,47, the negative regulator phosphatase triple mutant abi1-2/abi2-1/hab1-1 (pp2c 3 m)^48,49, and the key kinase mutant snrk2.2/2.3/2.6^50,51. We performed polysome profiling, which separates mRNA bound to different numbers of ribosomes using a sucrose gradient in ultracentrifugation²⁰. This analysis evaluated the global translation activity in Col-0 and the ABA signaling mutants, comparing mock treatment to 5 µM ABA treatment for 4 h of three-day-old seedlings (Fig. 2a–d). As previously reported¹¹, ABA treatment significantly reduces the polysome/monosome ratio in Col-0 compared to mock treatment, validating our polysome profiling approach (Fig. 2a).

**Fig. 2: ABA affects translation efficiency through its signaling pathway factors.**

In our polysome profiling analysis, the pyr1pyl1/2/4 mutant, which is less sensitive to ABA⁴⁷, showed no significant changes in the polysome/monosome ratio compared to Col-0 under mock condition (Supplementary Fig. 2). The pp2c 3 m mutant, known to be hypersensitive to ABA during seed germination and root growth⁴⁹, displayed a reduced polysome/monosome ratio compared to Col-0 under mock condition, similar to Col-0 treated with ABA (Fig. 2a, Supplementary Fig. 2). In contrast, the snrk2.2/2.3/2.6 mutant, which is almost completely insensitive to ABA and exhibits rapid water loss⁵¹, also demonstrated a reduced polysome/monosome ratio under mock condition (Supplementary Fig. 2). This water-sensitive state likely contributed to its decreased translation efficiency, as drought is known to inhibit mRNA translation⁵².

Notably, in response to ABA treatment, the polysome/monosome ratios in the pyr1pyl1/2/4, pp2c 3 m, and snrk2.2/2.3/2.6 mutants showed no significant changes compared to mock conditions in contrast to Col-0 (Fig. 2a–d), further confirming that core components of ABA signaling are essential for regulating the polysome profiles. To assess the role of ABA signaling components in regulating TE changes at specific genes, we quantified the down-regulation of the photosynthesis gene CEST and the abiotic stress response gene FRUCTOSE-BISPHOSPHATE ALDOLASE 2 (FBA2, At4g38970)⁵³ upon ABA treatment in different mutants using ribosome fractionation and qPCR. While Col-0 showed reduced TE for CEST and FBA2 in response to ABA, the pyr1pyl1/2/4, pp2c 3 m, and snrk2.2/2.3/2.6 mutants did not exhibit significant changes (Fig. 2e). The negative control PP2A showed no alterations in TE under ABA treatment compared to mock conditions in all tested plants (Fig. 2e).

Thus, ABA-mediated regulation of mRNA translation efficiency is accompanied by signal transduction from receptors to core signaling components, including phosphatases and kinases.

GRP7&8 is involved in ABA-mediated translational efficiency regulation

Given the changes in polysome profiling mediated by ABA signaling, we sought to identify factors associated with ribosomes that are regulated by the ABA signaling pathway. Immunoprecipitation followed by mass spectrometry (IP-MS) was used to identify these ribosome-associated factors. Using anti-Flag, we conducted IP on 3-days-old seedlings of pACTIN2::Flag-eL18y (see material for details), a component of the large ribosomal subunit¹⁷ (Fig. 3a, b), with Col-0 as a negative control. Co-immunoprecipitated proteins identified in both replicates from pACTIN2::Flag-eL18y but absent in Col-0 were considered associated factors with eL18y (Supplementary Data 3).

**Fig. 3: GRP7 is involved in ABA-mediated translation efficiency regulation.**

In addition to ribosomal subunits, mRNA binding proteins were enriched, including eight non-ribosomal RNA-binding proteins (RBPs) (Fig. 3c). Notably, GRP7 and COLD SHOCK PROTEIN 2 (CSP2, At4g38680) are RBPs that respond to ABA and exhibit altered sensitivity when mutated^54,55. Furthermore, a suite of mRNAs previously reported GRP7 mRNA targets from individual nucleotide resolution Crosslinking and Immunoprecipitation Sequencing (iCLIP-seq) data⁵⁶ significantly overlapped with genes showing upregulated or downregulated translation efficiency in response to ABA treatment in this study (Fig. 3d). Under ABA treatment, iCLIP-seq identified GRP7 targets with downregulated translation efficiency were primarily enriched in pathways related to photosynthesis, while targets with upregulated translation efficiency were mainly involved in water transport and channel activity (Fig. 3e). Collectively, these findings suggest that GRP7 may act as a key regulator in ABA-mediated translation regulation.

GRP8 closely resembles GRP7, displaying high sequence similarity and functional redundancy in regulating alternative splicing³⁴ and flowering⁵⁷. To further elucidate their roles in ABA-regulated plant development and potential involvement in mRNA TE regulation, we generated a grp7grp8 double mutant by editing the GRP8 gene in the grp7-1 background (Supplementary Fig. 3a). A single nucleotide insertion (A or G) in the first exon of GRP8 resulted in an altered reading frame with a stop codon at either the 8^th or 36^th amino acid position (Supplementary Fig. 3a). The seed germination of the grp7-1 mutant is hypersensitive to ABA⁵⁵. Notably, both grp7grp8^CR-A/G mutant lines exhibited significantly higher ABA sensitivity than either the grp7-1 or grp8^CR-A/G single mutants (Supplementary Fig. 3b), indicating the redundant functions of GRP7 and GRP8 in ABA-mediated inhibition of cotyledon greening.

We then performed the polysome profiling of Col-0 and grp7grp8^CR-A/G mutant lines under both mock and ABA treatments for 4 h. Notably, both grp7grp8^CR-A/G lines exhibited a reduced polysome/monosome ratio compared to Col-0 at mock condition (Fig. 3f, g), similar to the effect observed in Col-0 under ABA treatment (Supplementary Fig. 3c-top panel). When examining the polysome profiles during ABA treatment, both grp7grp8^CR-A/G lines showed lesser reduction in the polysome/monosome ratio, but ABA treatment still resulted in decreased ribosome loading efficiency in both mutant lines (Fig. 3h, i and Supplementary Fig. 3c–lower panel). This indicates that GRP7 and GRP8 partially mediate the effect of ABA on the global decline in mRNA translation efficiency. Additionally, the surface sensing of translation (SUnSET) assay (see "Methods"s for details) demonstrated that, under mock conditions, protein synthesis efficiency in grp7grp8^CR-A and grp7grp8^CR-G was lower compared to Col-0. Under ABA treatment, Col-0 displayed a decrease in protein synthesis efficiency, while almost no decrease was detected in the grp7grp8^CR-A and grp7grp8^CR-G lines (Fig. 3j, k). These results suggest that GRP7&8 plays a role in the regulation of translation in response to ABA.

ABA signaling regulates GRP7&8 level to participate in translation regulation

To further validate the association of GRP7&8 with the ribosomal machinery in the cytoplasm, we separated different mRNA-bound ribosome fractions using a sucrose gradient through ultracentrifugation of lysates from three-day-old pACTIN2::Flag-eL18y seedling treated with mock or 5 µM ABA for 4 h. Following this, we performed immunoblotting with various antibodies (Fig. 4a). We generated a GRP7 antibody based on a previous publication³⁷, which detect both GRP7 (16.9 KDa) and GRP8 (16.6 KDa) in Col-0, given their marginal size difference (Supplementary Fig. 4a). The Flag antibody was employed to detect Flag-eL18y, a component of the large ribosomal subunit, while eS6z marked the small ribosomal subunit¹⁷. GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT (GAPDH, At3g04120) served as a control protein not associated with ribosomes⁵⁸.

**Fig. 4: ABA signal reduces GRP7 protein to participate in translation regulation.**

As expected, Flag-eL18y was detected in the 60S large subunit, 80S monosome and polysome fractions, while eS6z was primarily found in the 40S small subunit, 80S monosome and polysome fractions under both mock and ABA treatment conditions (Fig. 4a), validating our procedure. GRP7&8 co-fractionated with the 40S and 60S ribosomal subunits, monosome, and polysome fractions (Fig. 4a), suggesting their active participation in the translation process. To rule out the possibility of non-ribosomal complexes co-fractionating, we treated the lysates with EDTA to disassemble 80S monosomes into 40S and 60S ribosomal subunits, which resulted in the depletion of GRP7&8 from the monosome and polysome fractions (Supplementary Fig. 4b–left). Additionally, treatment with RNase A, which promotes the accumulation of 80S monosomes, led to a substantial reduction in GRP7&8 levels within polysome fractions (Supplementary Fig. 4b–right). These findings robustly support the association between GRP7&8 and actively translating mature ribosomes.

Notably, under ABA treatment, the amount of GRP7&8 co-fractionating with polysome appeared reduced compared to mock conditions (Fig. 4a). Furthermore, a Co-IP assay confirmed that GRP7&8 interact with eL18y under both mock and ABA treatments (Fig. 4b). Importantly, this interaction was unaffected by ABA treatment when normalizing the GRP7 or GRP8 protein levels from IP sample to input samples (Fig. 4c). These results suggest that while ABA treatment does not influence the interaction between GRP7&8 and ribosomes, it likely affects their association with active translation polysomes.

We further examined the impact of ABA treatment on the expression of GRP7 and GRP8. RNA-seq data, confirmed by qPCR, revealed that the transcript levels of GRP7 and GRP8 significantly decreased in three-day-old seedling exposed to 5 µM ABA for 4 h (Fig. 4d), consistent with previous reports⁵⁵. qPCR analysis of entire fractions of polysome profiling also indicated a reduction in the TE of both mRNAs under ABA treatment (Fig. 4e), suggesting that their protein levels are likely diminished. Ribo-seq data revealed a notable decrease in the translational levels of GRP7 and GRP8 in Col-0 following ABA treatment (Supplementary Fig. 4c). Indeed, protein levels of GRP7 and GRP8 were significantly reduced in three-day-old seedlings treated with 5 µM ABA compared to mock (Fig. 4f, g). This reduction did not appear to be related to protein stability, as no differences in GRP7 and GRP8 levels was observed between mock and ABA treatments with the prior addition of cycloheximide (CHX) (Supplementary Fig. 4d), which inhibits new protein synthesis⁵⁹. Importantly, the decrease in GRP7 and GRP8 protein levels upon ABA treatment was abolished in core ABA signaling mutants such as pyr1pyl1/2/4, pp2c 3m and snrk2.2/2.3/2.6 (Fig. 4f, g). Thus, ABA signaling activation likely reduces GRP7 and GRP8 levels by reducing RNA levels and translation efficiency at the three-day-old seedling stage.

To genetically confirm the role of GRP7/8 in the ABA signaling pathway, we crossed snrk2.2/2.3/2.6 with grp7grp8^CR-G to create the homozygous quintuple mutant srnk2.2/2.3/2.6 grp7grp8^CR-G (5m) (Supplementary Fig. 4e). We assessed the germination rates of Col-0, snrk2.2/2.3/2.6, grp7grp8^CR-G, and 5 m on 1/2 MS medium with 0 μM, 1 μM, and 50 μM ABA, around 48 h post-stratification. As reported^35,51, snrk2.2/2.3/2.6 was resistant while grp7grp8^CR-G was sensitive to ABA compared to Col-0 (Fig. 4h, i, Supplementary Fig. 4f, g). Under low ABA concentration (1 μM), the sensitivity of 5 m matched that of snrk2.2/2.3/2.6 (Supplementary Fig. 4f, g). However, at high ABA concentration (50 μM), 5 m was more sensitive than snrk2.2/2.3/2.6 (Fig. 4h, i), indicating GRP7&8’s role in mediating morphological responses to elevated ABA levels.

GRP7&8 mediates ABA signaling-regulated mRNA translation efficiency

To assess the role of GRP7&8 in translation efficiency regulation, we performed Ribo-seq and RNA-seq on grp7grp8^CR-A with mock and ABA treatment for 4 h. Firstly, quality assessment using principal component analysis revealed a high degree of reproducibility among three biological replicates of RNA-seq and Ribo-seq data (Supplementary Fig. 5a). Next, we analyzed the features of the ribosome footprint revealed by Ribo-seq reads. We found that the most abundant Ribo-seq read lengths were 27 and 28 nt (Supplementary Fig. 5b), and the Ribo-seq reads mapped to 3’ UTR were significantly decreased compared to RNA-seq reads (Supplementary Fig. 5c). Notably, the distribution of Ribo-seq 5’ end counts across CDS displayed 3-nt periodicity pattern and Ribo-seq 5’ end counts were enriched at 15-18 nt upstream of start codon and stop codon (Supplementary Fig. 5d–g). Notably, second peaks were found around start codon, which have been consistently observed in many ribosome profiling datasets due to ligate sequence preference^60,61. Overall, these features indicate the high quality of these Ribo-seq profiling data. As expected, the abundance of RNA-seq read mapped to each gene were strongly correlated with the abundance of Ribo-seq reads within the same sample (Supplementary Fig. 6).

In grp7grp8^CR-A, the mRNA abundance levels of 759 and 1087 genes were significantly changed compared to Col-0 under mock and ABA treatment, respectively (Fig. 5a and Supplementary Data 4, 8). However, at the translational level, 2062 genes and 2077 genes displayed altered TE in the grp7grp8^CR-A under normal conditions and ABA treatment, respectively (Fig. 5b, Supplementary Data 6, 9). These results indicate that GRP7&8 affects more genes through translational regulation than at the mRNA level. Under mock condition, 250 genes exhibited changes at both mRNA and translational level in grp7grp8^CR-A compared to Col-0. Additionally, 509 genes displayed altered mRNA level but maintained unchanged TE, while 1,812 genes changed TE while their mRNA level remained constant (Fig. 5c). Remarkably, genes exhibiting ABA-repressed TE in Col-0 significantly overlapped with genes displaying reduced TE in grp7grp8^CR-A compared to Col-0 under normal condition. (Fig. 5d). Moreover, the heatmap of genes with altered TE in both Col-0 under ABA treatment and in grp7grp8^CR-A under mock conditions revealed that the majority of genes (73.68%) exhibited similar patterns of TE change in ABA treatment and the grp7grp8^CR-A mutation. Among them, most genes showed a significant down-regulation in TE, while a subset of genes exhibited up-regulation in TE (Fig. 5e).

**Fig. 5: ABA-regulated global translational efficiency dependent on GRP7&8.**

To further explore the effect of GRP7&8 on mediating ABA-triggered mRNA and translation efficiency level changes, we performed a comprehensive analysis between genes regulated by GRP7&8 and ABA-responsive genes. Under ABA treatment, 4610 DEGs and 2584 genes with DTEs were found in grp7grp8^CR-A compared to mock, respectively (Fig. 5f and Supplementary Data 5, 7). At mRNA level, we found that, among the 5,248 ABA-induced differentially expressed genes in Col-0, 2,118 (~40.35%) genes changed their responses in grp7grp8^CR-A mutant plant (Fig. 5g, h and Supplementary Data 1, 5), for detailed data process, see "Methods"). At translational level, among the 1548 genes with ABA-regulated TE alteration in Col-0, 1255 ( ~ 81.07%) genes were disrupted in grp7grp8^CR-A mutant (Fig. 5i, j, Supplementary Data 2, 7). Notably, among those genes with TE changed in GRP7&8-dependent and ABA-responsive manner, genes with ABA-repressed TE enriched in GO terms related to photosynthesis and chlorophyll biosynthesis, while genes with ABA-promoted TE tent to enrich in response to ABA and response to water deprivation (Fig. 5k). Using polysome profiling followed by RT-qPCR, we further measured the TE of photosynthesis gene CAB2, stomatal regulation gene BETA CARBONIC ANHYDRASE 2 (CA2, At5g14740)⁶², and abiotic stress-responsive gene FBA2 upon ABA treatment in Col-0 and grp7grp8^CR-A/G mutants, confirming that their down-regulation of TE after ABA treatment were GRP7&8 dependent (Fig. 5l). This comprehensive analysis establishes GRP7&8 as key mediators of the ABA-regulated translational response.

GRP7&8 mediates translational regulation mainly by directly binding to targets

To explore whether GRP7&8 mediates the translational efficiency regulation of ABA signaling by binding to its target mRNAs, we performed protein-RNA crosslinking and immunoprecipitation (CLIP)-seq of GRP7-GFP coupled with GFP-Trap⁶³ (Fig. 6a and Supplementary Fig. 7a, b). RNA-protein complexes were immunoprecipitated with GFP Trap beads and bound RNAs were used for constructing CLIP-seq libraries (Supplementary Fig. 7a, b). Most of the GRP7 targets were enriched at 5’ UTRs and exons of protein-coding mRNAs (Fig. 6b, Supplementary Fig. 7c, Supplementary Data 10). Totally, 2468 and 3514 GRP7-binding genes were identified under normal and ABA conditions, respectively. We found that the target mRNAs of GRP7 with mock and ABA treatment significantly overlap with the previously reported GRP7 target mRNAs⁵⁶ (Supplementary Fig. 7d). Overall, 590 (68.76%) out of the 858 known GRP7-binding targets were identified in our CLIP-seq datasets, indicating high-confidence of the identified GRP7 targets. Furthermore, the motifs of GRP7 binding peaks were U/C enriched (Supplementary Fig. 7e), which were consistent with previous report⁵⁶.

**Fig. 6: GRP7/8 mediates translational regulation of ABA signaling by binding to its target mRNAs.**

Importantly, we found a significant overlap between the target mRNAs of GRP7 under both mock and ABA treatment conditions and the DTE altered by ABA treatment (Fig. 6c). Of the 1548 DTE responsive to ABA, 716 (46.25%) were identified as GRP7-binding targets, which motifs were also U/C enriched (Supplementary Fig. 7e). GO enrichment analysis revealed that genes related to abiotic stress and photosynthesis were enriched among GRP7 target mRNAs that were bound under mock conditions or under both mock and ABA conditions, but not among those bound solely under ABA conditions (Fig. 6d). Notably, the translational efficiency of GRP7 target mRNAs significantly decreased in the grp7grp8^CR-A mutant compared to Col-0, under both mock and ABA conditions (Fig. 6e). The genes with altered translation efficiency due to GRP7-mediated ABA signaling, including the photosynthesis gene CAB2, the stomatal regulation gene CA2, and the abiotic stress-responsive gene FBA2, were all direct targets of GRP7 identified through CLIP-seq data (Fig. 6f). Further validation using RIP-qPCR confirmed the binding of GRP7 to these genes, while the non-target mRNA RGS1-HXK1 INTERACTING PROTEIN 1 (RHIP1, At4g26410) served as a negative control (Fig. 6g). Collectively, these results suggest that GRP7 mediates the regulation of mRNA translation efficiency in response to ABA signaling through its binding to target mRNAs.

Drought regulated translation partly through ABA-GRP7&8

Drought stress induces ABA accumulation and activates ABA-dependent and independent pathways that modify plant morphology for adaptation⁴³. Research indicates that drought can trigger global translational changes^42,64. We found both the GRP7-binding targets and genes with GRP7/8-depdenent DTE were enriched in the GO term response to water deprivation (Figs. 5k, 6d), indicating a potential role of GRP7 in plant drought response. To investigate whether drought regulates translational changes through the ABA-GRP7&8 pathways, we performed polysome profiling in Col-0, the ABA biosynthetic enzyme mutant aba2-1⁶⁵, and grp7grp8^CR-A/G mutants under PEG treatment for 4 h. PEG treatment significantly reduced the polysome/monosome ratio in Col-0, but this reduction was notably less pronounced in the aba2-1 and grp7grp8^CR-A/G mutants (Fig. 7a and Supplementary Fig. 8), indicating that drought-induced polysome profiling pattern alteration is partially dependent on the ABA-GRP7&8 pathway.

To assess the biological function of GRP7&8-mediated translational regulation under drought stress, we examined the drought tolerance phenotype of the grp7grp8^CR-A mutant. This mutant exhibited enhanced drought tolerance compared to Col-0, with a significantly higher survival rate following drought exposure (Fig. 7b, c) and a reduced water loss rate in detached leaves (Fig. 7d). Furthermore, we evaluated the translation efficiency of FBA2, a gene associated with abiotic stress response⁵³, in Col-0, aba2-1, and grp7grp8^CR-A/G mutants under control and PEG treatment (Fig. 7e). In Col-0, PEG treatment significantly reduced FBA2 translation efficiency, whereas it remained relatively unchanged in the aba2-1 and grp7grp8^CR-A/G mutants (Fig. 7e). Given that FBA2 is a target of GRP7 (Fig. 6g), these findings suggest that the drought-induced changes in its translation efficiency are partially regulated by the ABA-GRP7&8 pathways (Fig. 7f).

Discussion

Plants, being sessile organisms, constantly face various abiotic stresses. To adapt, they have developed intricate strategies, including precise transcriptional networks that sense harsh environmental cues and redirect developmental programs⁶⁶. Additionally, translational regulation is a crucial pathway that enhances plant growth plasticity^14,42. Despite its importance, the signaling mechanisms linking abiotic stress to mRNA translation remain largely unexplored¹³. In this study, we focus on ABA signaling to address this challenge.

ABA is a crucial phytohormone involved in plant growth and stress responses^1,5. Our research reveals that ABA treatment not only influences mRNA level but also significantly affects mRNA translation efficiency (Fig. 1). Specifically, ABA inhibits global mRNA translation via its core signaling pathway, which includes the receptors PYR/PYL/RCARs, the phosphatase PP2Cs, and the kinase SnRK2s (Fig. 2). Notably, the snrk2.2/2.3/2.6 mutants exhibit a reduced polysome/monosome ratio compared to Col-0 under mock conditions, likely due to their highly water-sensitive state⁵¹, as drought is also known to inhibit translation⁵². Additionally, a decreased polysome/monosome ratio was observed from two to four days after germination (Supplementary Fig. 9a, b), indicating that early developmental stages significantly influence global translation efficiency. Mutations in pp2c 3 m and snrk2.2/2.3/2.6 affected early developmental pace and morphology, while pyr1pyl1/2/4 mutants showed no significant changes (Supplementary Fig. 9c). The alterations in the polysome/monosome ratio in pp2c 3 m and snrk2.2/2.3/2.6 may reflect developmental differences in addition to the direct effects of ABA signaling under mock conditions. Future studies could explore how endogenous developmental cues interact with exogenous stimuli to coordinate the regulation of mRNA translation.

While ABA signaling primarily influences transcription through AREB/ABF transcription factors¹, it also modulates translation efficiency via specific RNA-binding proteins, particularly GRP7&8. ABA treatment reduces GRP7&8 protein levels, a process dependent on signaling transduction from PYR/PYL/RCARs to SnRK2s (Fig. 4f, g). Interestingly, the grp7grp8^CR-A/G mutants exhibit a more sensitive physiological response to ABA, such as inhibited germination, compared to Col-0 (Supplementary Fig. 3b). This suggests that, even when the translational response is disrupted in grp7grp8^CR-A/G mutants, normal transcriptional regulation still occurs. To explore how these two regulatory arms coordinate, we assessed the germination phenotypes of Col-0, snrk2.2/2.3/2.6, grp7grp8^CR-G, and the quintuple mutant (5 m) under varying ABA concentrations (0, 1, and 50 µM) (Fig. 4h, i and Supplementary Fig. 4f, g). Under mock (0 µM) and low ABA concentrations (1 µM), transcriptional regulation via AREB/ABFs appears to drive physiological responses, as the 5 m mutant phenocopied the snrk2.2/2.3/2.6 phenotype. In contrast, at high concentrations (50 µM), GRP7&8-mediated translational regulation becomes more influential, evidenced by the increased sensitivity of the 5 m plants compared to snrk2.2/2.3/2.6. Overall, ABA signaling operates at both mRNA and translational levels, potentially through distinct pathways downstream of SnRK2s, adapting plant responses to varying ABA concentrations (Fig. 7f).

Upon receiving developmental or environmental signals, translation regulators modulate mRNA translation efficiency, leading to either global reprogramming or gene-specific responses⁶⁷. Under elevated temperatures, GRP7 rapidly forms condensates that recruit target mRNA and components of the translation machinery, such as eukaryotic initiation factor 4E1 (eIF4E1) and cold shock proteins 1 (CSP1) and 3 (CSP3), thereby inhibiting the formation of translation complexes and the translation of heat stress genes⁴¹. In immune responses, GRP7 associates with an active translational complex linked to phosphorylated eIF4E at the mRNA cap, potentially regulating the translation of specific defense-related genes⁴⁰.

In this study, we found that GRP7&8 co-fractionate with the 40S and 60S ribosomal subunits, monosomes, and polysomes, indicating their involvement in maintaining proper translation efficiency for developmental genes in three-day-old seedlings, including those related to photosynthesis and chloroplast biogenesis (Fig. 5k). Following ABA treatment, GRP7&8 protein levels decreased, along with their association with polysomes (Fig. 4), resulting in a decline in global mRNA translation efficiency. The absence of GRP7 and GRP8 significantly disrupts ABA-responsive translation efficiency, with a large portion of DTE affected, highlighting ABA’s regulation of translation through GRP7 and GRP8 (Fig. 5). However, ABA treatment still reduces the polysome/monosome ratio in the absence of GRP7&8 (Fig. 3h, i), suggesting that ABA regulates translation through both GRP7&8-dependent and -independent pathways (Fig. 7f). These pathways may function sequentially, with the GRP7&8-independent mechanism initiating the first wave for the translational and transcriptional regulation, including GRP7&8 expression itself, while decreased GRP7&8 levels trigger a secondary wave of translational regulation (Fig. 7f).

Furthermore, the translation efficiency of GRP7-binding targets significantly decreases in plants lacking GRP7&8, suggesting that GRP7&8’s regulation of translation efficiency relies on its direct binding to target mRNAs (Fig. 6e). Notably, GRP7 associates with ribosomes even in the absence of RNA binding (Fig. 4a, b and Supplementary Fig. 4b), suggesting that GRP7 may facilitate target mRNA translation by promoting polysome loading. Unlike heat stress, where GRP7 specifically targets several mRNAs, ABA treatment induces a broader reprogramming of mRNA translation efficiency through GRP7&8. Additionally, GRP7’s regulation of specific genes may involve different mechanisms, promoting some developmental genes while inhibiting stress-related genes under normal condition.

Interestingly, ABA treatment reduces GRP7&8 protein levels, not through alterations in protein stability, a finding consistent with previous reports³⁵. Our study shows that treatment with 5 µM ABA in three-day-old seedlings suppresses GRP7&8 mRNA level, as demonstrated by RNA-seq and RT-qPCR analyses (Fig. 4d). This aligns with findings in two-week-old plants treated with 100 µM ABA⁵⁵ but contrasts with reports of increased GRP7 mRNA levels in 7-day-old plants under 100 µM ABA³⁵. Previous studies indicated that ABA treatment elevates levels of alternative spliced GRP7 (as_GRP7), which contains a premature stop codon, while decreasing levels of fully spliced GRP7 RNA (fs_GRP7)³⁵. Our results reveal a decline in both as_GRP7 and fs_GRP7 levels in three-day-old seedlings treated with 5 µM ABA (Supplementary Fig. 10), alongside a decrease in GRP7 translation efficiency (Fig. 4e). We speculate that variations in developmental stages and ABA concentrations may lead to differential regulation of GRP7 by ABA treatment. Further investigation into how ABA regulates GRP7 and GRP8 from multiple perspectives would be valuable.

Given that abiotic stresses like drought and salinity can trigger ABA biosynthesis and influence plant development^5,6, we used drought as a model to explore its effects. Our findings suggest that drought partially relies on the ABA-GRP7&8 modules to modulate mRNA translation. Translational regulation responds more rapidly to environmental stresses than transcriptional regulation, enabling swift adaptation⁶⁸. Thus, investigating the comparative roles of transcription and translation regulation in the rapid response and long-term adaptation to drought through transcriptome and translatome analyses at different time points will be an intriguing area for further study.

Methods

Plant materials and growth conditions

Arabidopsis thaliana plants used in this study were in the Col-0 background. The seeds were sterilized using a 75% ethanol solution with 0.1% Triton X-100. After sterilization, they were sown onto plates with half-strength Murashige and Skoog (MS) medium, which had been adjusted to a pH of 5.7 and supplemented with 1% (w/v) sucrose and 0.8% (w/v) agar. These plates were then placed in a dark cold room for stratification at 4 °C for a period of three days. Following this, the plates were moved to a light incubator under long-day conditions (LD, 16 h of light and 8 h of darkness) at 22 °C, and subject to mock, ABA or PEG treatment at ZT10. For ABA treatment, three-day-old seedlings were transferred onto filter paper that had been moistened with 1/2 MS liquid medium containing 5 μM ABA or an equivalent volume of mock solution. They were then incubated under the same long-day conditions at 22 °C for 4 h. For PEG treatment, 6-days-old seedlings were transferred onto filter paper that had been moistened with 1/2 MS liquid medium containing 15% PEG or an equivalent volume of mock solution. They were then incubated under the same long-day conditions at 22 °C for 4 h. The seedlings were then gently dried and flash-frozen in liquid nitrogen. The samples were stored at -80 °C for further analysis.

To generate pACTIN2::FLAG-eL18y, the eL18y CDS sequence was amplified by PCR using cDNA as a template. The resulting DNA fragment was ligated into the pCAMBIA3301 vector driven by the ACTIN2 promoter from Arabidopsis thaliana. The resulting construct was transformed into Agrobacterium tumefaciens GV3101 and transferred to Col-0 plants using the floral dip method. The grp7grp8^CR-A, grp7grp8^CR-G, grp8^CR-A and grp8^CR-G mutants were created by CRISPR/Cas9 editing. grp7-1 (SALK_039556) mutant described previously⁷. The pp2c triple mutant (3 m) (abi1-2abi2-2hab1-1)⁴⁸, snrk2.2/2.3/2.6⁵⁰, aba2-1⁶⁹ and pyr1pyl1/2/4⁴⁶ were described previously. The pGRP7::GRP7-GFP grp7-1 complementary line⁵⁶ was described previously. The snrk2.2/2.3/2.6 grp7grp8^CR-G quintuple mutant (5 m) was established by crossing snrk2.2/2.3/2.6 and grp7grp8^CR-G. The primers used for identification of the mutations are listed in Supplementary Data 11.

Physiological experiments

For the germination assay⁷⁰, approximately 50 seeds were sown on 1/2 MS medium supplemented with varying concentrations of ABA. The plates were initially incubated at 4 °C for 3 days and then transferred to a light incubator under LD conditions for a subsequent 15-day growth period to assess seed germination rates. Photographs were taken continuously throughout this period. Seed germination was defined as the radicle rupture of the seed coat. Green cotyledons were defined as seedlings with expanded cotyledons. The experiment was performed with three replicates.

For the drought stress tolerance⁴⁸, 7-days-old seedlings were transplanted into pots and cultivated under normal long-day growth conditions for a period of two weeks. Subsequently, drought stress was imposed by withholding water from the seedlings for an additional two weeks. The drought phenotypes were photographed three days following rehydration. To assess water loss in detached leaves, 7-day-old seedlings were transplanted into soil and cultivated for 3 weeks under a 16-h light/8-h dark photoperiod. The fifth true leaves from plants grown under same conditions were selected. The leaves were cut, placed on weighing paper and weighted immediately. Four leaves were used for each genotype and placed on the same piece of weighing paper. The leaves were then returned to normal growth conditions. The relative water loss rate was calculated as the ratio of water loss to the initial fresh weight.

Polysome profiling analysis

Arabidopsis thaliana polysomes were fractionated over sucrose gradients as described with minor modifications¹⁹. In brief, three-day-old seedlings were treated with 5 μM ABA for 4 h and then ground in liquid nitrogen followed by resuspension in polysome extraction buffer containing 200 mM Tris-HCl pH 9.0, 200 mM KCl, 35 mM MgCl₂, 25 mM EGTA, 1% sodium deoxycholate, 1% Detergent mix (20% Briji, 20% Triton X-100, 20% Igepal CA630, 20% Tween 20), 1% Polyoxyethylene 10 tridecyl ether (PTE), 5 mM DTT, 1 mM PMSF, 50 μg/mL cycloheximide, 50 μg/mL chloramphenicol, and 80 U/mL SUPERase-In RNase Inhibitor (Invitrogen Cat# AM2694). The supernatant was loaded onto a 10%–50% sucrose gradient (10 × Sucrose salt buffer: 400 mM Tris-HCl pH: 8.4, 200 mM KCl, 100 mM MgCl₂) and spun in a Beckman SW41Ti rotor at 33,500 rpm for 3 h at 4 °C. We collected 11 fractions by a gradient fractionator. For RNase A treatment⁷¹, SUPERase-In RNase Inhibitor was omitted from the polysome extraction buffer. After lysis, 1 μL of RNase A (Invitrogen, catalog no. AM2270) was added to the lysate. For EDTA treatment⁷¹, lysates were layered on a linear sucrose gradient (10%–50% sucrose (w/v), 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM EDTA, and 1 mM DTT).

For polysome RNA extraction¹⁹, 600 μL of phenol–chloroform–isoamyl alcohol were pipetted into a 600 μL sample of different fractions. Then, 15 μL of 10% SDS, 12 μL of 0.5 M EDTA (pH 8.0), and 3 μL of 1 M DTT were added to each tube and mixed thoroughly. The samples were then extracted and centrifuged at 12,000 g for 15 minutes. The supernatant (500 μL) was transferred to a fresh tube. RNA was precipitated by adding 50 μL of 3 M sodium acetate (pH 5.2) and 500 μL of isopropanol. The RNA was subjected to reverse transcription and qPCR analysis.

For polysome protein extraction, we added 900 μL of methanol, 220 μL of trichloromethane, and 600 μL of H₂O to a 600 μL sample of different fractions. The mixture was then well mixed and allowed to stand for 10 minutes at room temperature before being centrifuged at 18,000 g for 5 minutes at room temperature. After carefully removing the upper aqueous phase, 600 μL of methanol were added to each tube, mixed well, and centrifuged again at 18,000 g for 5 min at room temperature. Finally, 50 μL of protein loading buffer was added to the precipitate and incubated at 95 °C for 10 minutes. The sample is now ready for subsequent experiments, such as Western blotting.

SUnSET assay

SUnSET assay is modified from a previous protocol²⁹. Briefly, three-day-old seedlings were treated with mock and 5 μM ABA for 3.5 h, after which 50 μM puromycin was applied to the samples for 30 minutes. The seedlings were ground in liquid nitrogen, to which 2× SDS loading buffer was subsequently added. After vigorous mixing, the lysates were boiled at 95 °C for 10 minutes. Puromycin-labeled proteins were detected by western blot probed with an anti-puromycin antibody (EMD Millipore, MABE343, 1:3000 dilution).

RNA immunoprecipitation (RIP) and qPCR analyses

RIP was performed as described previously with some modifications⁷². One gram of three-day-old seedlings were treated with mock and 5 μM ABA for 4 h and then ground in liquid nitrogen followed by crosslinking twice at 600 mJ/cm² in a UVP crosslinker (Analytik jena). Samples were solubilized using 2 mL of extraction buffer, composed of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 4 mM MgCl₂, 0.1% Igepal CA-630, 5 mM DTT, 0.1% SDS, 1 mM PMSF, protease inhibitor, and 80 U/mL SUPERase-In RNase Inhibitor. 100 μL supernatant was used as input. RNA-GRP7-GFP complexes were enriched from the supernatant by immunoprecipitation with GFP-trap beads for 2 h at 4 °C under constant rotation. The GFP-Trap beads (Lablead, GNM-25-1000) were then washed four times with a washing buffer containing 50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Igepal CA-630, and 0.1% SDS. To elute the protein–RNA complexes, the beads were incubated at room temperature for 10 minutes with gentle rotation in 50 μL of RIP elution buffer, which contained 100 mM Tris-HCl at pH 7.4, 100 mM NaCl, 10 mM EDTA, 1% SDS, and 80 U/mL RNase inhibitor. The protein was degraded by proteinase K, and co-precipitated RNAs were eluted by 1 mL TRIzol of RNA extraction reagent. The RNA sample was incubated with DNase I and reverse-transcribed using cDNA synthesis kit (TIANGEN, KR116) for qPCR. In parallel, input samples were used for quantification. The primers used for qPCR are listed in Supplementary Data 11.

Ribosome profiling (Ribo-seq)

Ribosome profiling experiment was performed as previous reported with some modifications^73,74. In brief, three-day-old seedlings were treated with 5 μM ABA for 4 h and then ground in liquid nitrogen followed by resuspension in 600 μL ice-cold lysis buffer. Upon clarifying the lysate by centrifugation for 10 minutes at 20,000 g at 4 °C, the soluble supernatant was recovered. 6 μL of RNase I (100 U/μL) were added to 600 μL lysate and incubated for 45 minutes at room temperature with gentle mixing. 10 μL SUPERase-In RNase Inhibitor was then added to stop nuclease digestion. Meanwhile, MicroSpin S-400 HR columns (GE Healthcare Cat# 275140-01) were equilibrated with 3 mL of mammalian polysome buffer by gravity flow and emptied by centrifugation at 600 g for 4 minutes. The digested lysate was immediately loaded on the column and eluted from the column by centrifugation at 600 g for 2 minutes. The RNA was extracted from the flow-through using Trizol (Thermo Fisher, 15596018CN). The ribosomal RNA fragments were removed using the Ribo-off rRNA depletion kit (Plant) (Vazyme, N409) and separated on a 15% denaturing urea-PAGE gel. The size ranges from 27 nt to 30 nt was cut and thus obtained RNA fragments were subjected into library generation using Smarter smRNA-Seq kit (Takara Cat# 635031).

Crosslinking and Immunoprecipitation Sequencing (CLIP-seq)

CLIP was performed as previously described with minor modifications^63,72. In brief, three-day-old seedlings were treated with mock and 5 μM ABA for 4 h and seedling samples (approximately 1 g) were harvested. Seedling samples were then rapidly frozen in liquid nitrogen and ground into a fine powder, which was crosslinked twice with 254 nm ultraviolet light at a dose of 600 mJ/cm² in the mortar pre-cooled with liquid nitrogen. The sample powder was suspended in lysis buffer, treated with DNase I. The resulting lysate was cleared by centrifugation, after which immunoprecipitation was performed using GFP-Trap beads. After immunoprecipitation, the RNA-protein complex was washed extensively several times with high-salt wash buffer and partially digested by micrococcal nuclease (2 × 10⁻⁵ U/μL, Takara, 2910 A)²⁷. The digested RNA was treated with polynucleotide kinase to remove the 3ʹ-terminal phosphate from RNA. The immunoprecipitated RNAs were ligated with RNA adaptors (biotin labeling) and the resulting complex was eluted using SDS loading buffer. Next, 10% of the eluate was separated on a 4–12% NuPAGE (Thermo Fisher) gel and transferred to a nitrocellulose membrane for biotin staining to assess enrichment of the RNA bound by target protein. The remaining 90% of the eluate was run in parallel. The nitrocellulose membrane was excised and RNA was released from the complex by proteinase K treatment. RNA was further purified and reverse-transcribed using AffinityScript reverse transcriptase (Agilent) with a primer complementary to the RNA adaptor to produce complementary DNA. The cDNA was purified and a DNA adapter was ligated to the 3’ end. The final library was amplified from the cDNA by Q5 HotStart DNA polymerase using primers specific to adaptors. The resulting PCR products were purified and resolved on a 3% agarose gel, and fragments in the size range of 150 to 200 bp were recovered from the gel and used for paired-end Illumina (PE150) sequencing.

Immunoprecipitation Mass Spectrometry (IP-MS) assay

Immunoprecipitation of Flag-eL18y proteins from three-day-old pACTIN2::FLAG-eL18y and Col-0 (negative control) seedlings was performed as previously described with some modifications⁷¹. Proteins were solubilized from plant powder using buffer A (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCl₂, 1 mM DTT, 8% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate, 100 μg/mL Cycloheximide (CHX), 100 U/mL SUPERase-In RNase Inhibitor, 25 U/mL Turbo DNase, Protease Inhibitor). Anti-FLAG M2 magnetic beads (Sigma-Aldrich, Cat#M8823) were used to immunoprecipitate Flag-eL18y from the protein extracts. IP samples were first washed three times for 5 minutes each at 4 °C with buffer B (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCl₂, 1 mM DTT, 1% Triton X-100, 0.5% sodium deoxycholate, 100 μg/mL CHX). Afterward, the beads were washed three times for 5 minutes each at 4 °C using buffer C (25 mM Tris-HCl pH 7.5, 300 mM NaCl, 15 mM MgCl₂, 1 mM DTT, 1% Triton X-100, 0.5% sodium deoxycholate, 100 μg/mL CHX). The resulting complex was eluted using SDS loading buffer. The Coomassie-stained gel strips were subjected to destaining, followed by reduction with DTT and alkylation with iodoacetamide. After these treatments, the gel strips were digested with trypsin overnight. Subsequently, the peptides were extracted in multiple steps using different concentrations of acetonitrile, which subjected to MS analyses by Liquid Chromatography–Mass Spectrometry (LC-MS, nanoLC-Q EXACTIVE, Thermo Scientific). There were two biological replicates for both the experimental group and the control group.

Western blot assay

For western blot analysis, protein samples were resolved by SDS-PAGE and then transferred onto a PVDF membrane. The membrane was blocked with 5% milk in PBST (containing 0.1% Tween-20) for 1 hour at room temperature. Subsequently, the membrane was incubated with primary antibodies: anti-GRP7 (1:2000 dilution), anti-Flag (Sigma, Cat#F1804, 1:5000 dilution), anti-eS6z (PHYTOAB, Cat#PHY2025A, 1:2000 dilution) antibody and anti-GAPDH (Proteintech, Cat#60004, 1:5000). Anti-actin antibody (LabLead, Cat#BP0101, 1:5000 dilution) was used as internal control. Immunoblotting was done by using the enhanced chemiluminescence (ECL) system.

Bioinformatics analysis

Raw reads of RNA-seq, Ribo-seq, and CLIP-seq were filtered by fastp v0.20.1 for adapters removing, low-quality bases trimming, and reads filtering⁷⁵. RNA-seq reads were filtered with parameter “--detect_adapter_for_pe”, CLIP-seq reads were filtered with parameter “--adapter_sequence TGGAATTCTCGG --adapter_sequence_r2 GATCGTCGGACT”. For Ribo-seq data, only first sequencing reads (*_R1.fq.gz) were used and filtered with parameter “-a AAAAAAAAAA -f 3 -l 16”. The high-quality reads of RNA-seq were mapped to the Arabidopsis thaliana genome (TAIR10) using STAR (v2.7.10) with default parameters⁷⁶. The clean reads of Ribo-seq were firstly aligned against the non-coding RNA sequences of Arabidopsis thaliana downloaded from Ensembl Plants⁷⁷ using bowtie2 (ref. ⁷⁸) to produce the unaligned reads. The unaligned reads were mapped to reference genome using STAR with parameters “--outFilterMismatchNmax 2 --outFilterMultimapNmax 1 --outFilterMatchNmin 14 --alignEndsType EndToEnd”. For CLIP-seq data, the clean reads were mapped to reference genome using STAR with no more than two mismatches⁷⁶ and the duplicates in mapped reads were removed using Picard v2.23.3. The overview of RNA-seq, Ribo-seq, and CLIP-seq total reads, filtered read and mapped reads is summarized in Supplementary Data 12.

Two or three replicates bam files were merged using Samtools v1.4 (ref. ⁷⁹). To normalize and visualize the individual and merged replicate datasets, the BAM files were converted to bigwig files using bamCoverage provided by deepTools v3.3.0 with 1 bp bin size and normalized by RPKM (Reads Per Kilobase per Million mapped reads) with parameters “-bs 1 --effectiveGenomeSize 120,000,000 --normalizeUsing RPKM --smoothLength 5” (ref. ⁸⁰). The number of reads that mapped to each gene was counted using featureCounts v2.0.1 with the parameter “-p -P -B -C” for RNA-seq and default parameters for Ribo-seq⁸¹. The raw counts were further normalized to FPKM (Fragments per kilobase per million mapped reads) for RNA-seq and RPKM (Reads per kilobase per million mapped reads) for Ribo-seq. FPKM or RPKM values of genes were Z-scaled and clustered by k-means method and displayed using R package ComplexHeatmap (v2.4.3) (ref. ⁸²). Gene Ontology enrichment was performed using an R package clusterProfiler v3.18.1 (ref. ⁸³).

The raw counts files of RNA-seq were used as inputs for differential expression analysis by DESeq2 v1.26.0 (ref. ⁸⁴). We quantified the mapped reads of RNA-seq and Ribo-seq across the 5’ UTR, CDS, and 3’ UTR regions, then calculated the percentage of reads in each region to determine the distribution ratio of reads in the 5’ UTR, CDS, and 3’ UTR. The Ribo-TISH toolkit was employed to analyze the distribution of Ribo-seq 5’ end counts across the three reading frames within the CDS of all genes, as well as the distribution of Ribo-seq 5’ end counts around the start and stop codons of all annotated genes. Only reads located within coding sequences (CDSs) were retained for translation efficiency analyses. Translation efficiency was calculated using the Xtail package (v1.1.5), which employs a Negative Binomial (NB) model to estimate the distributions of both mRNA and ribosome-protected mRNA fragments (RPF)⁸⁵. The analysis includes an evaluation of both the statistical significance and the magnitude of differential translation for each gene. Differential translation efficiency was identified using a threshold of P.adjust <0.05 (Supplementary Data 1, 2, 4-9).

At the mRNA level, 1,018 down-regulated and 1,100 up-regulated genes under ABA treatment in Col-0, which lost their responses in grp7grp8^CR-A were defined as GRP7-dependent ABA-responsive genes. A total of 2,118 out of 5,248 genes (40.35%) showed GRP7-dependent responses under ABA treatment (Fig. 5g, h). Similarly, 657 genes with down-regulated DTE and 598 genes with up-regulated DTE under ABA treatment in Col-0, which lost their responses in grp7grp8^CR-A, were defined as GRP7 dependent ABA-responsive genes at translational level. A total of 1,255 out of 1,548 genes (81.07%) showed GRP7-dependent DTE responses under ABA treatment (Fig. 5i, j).

The GRP7 binding sites were identified by performing CLIP-seq peak calling using CLIPper (v0.1.4) with default parameters⁸⁶. Then, bedtools was used to retain the peaks that were identified in both biological replicates⁸⁷. Finally, we defined the peaks that were only identified in IP samples but depleted in negative controls as significant binding sites of GRP7. The GRP7 binding peaks were annotated to the Arabidopsis thaliana genome using the “annotatePeak” function in R package ChIPseeker (v1.26.2) (ref. ⁸³) (Supplementary Data 10). The sequences from GRP7 binding peaks were extracted from genome using Biopython module. The motif enrichment analysis of the GRP7 binding sequences were performed using the online MEME Suite (https://meme-suite.org/meme/doc/overview.html).

Statistics & Reproducibility

R (https://cran.r-project.org/;version4.0.2) was used to compute statistics and generate plots if not specified. The Integrative Genomics Viewer (IGV) was used for the visual exploration of genomic data⁸⁸. The intersection between two sets of genes was displayed using the R package VennDiagram v1.7.3. One-sided Hypergeometric test was used in Figs. 1d, 3d, 3e, 5k, 6c, 6d, Supplementary Figs. 1b, 7d. The Pearson correlation coefficient analysis was used in Supplementary Figs. 1a, 6. The two-sided student’s t-test was used in Figs. 1f, 2a–e, 3g, 3i, 4c–e, 4g, 5l, 6g, 7a, 7c–e, Supplementary Figs. 2b, 3b, 3c, 8. Two-way ANOVA was used in Figs. 3k, 4i, Supplementary Fig. 4g. Two-sided Wilcoxon rank-sum test was used in Fig. 6e. Tukey’s HSD multiple comparison test was used in Supplementary Fig. 9b. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The raw sequence data of RNA-seq, Ribo-seq, and CLIP-seq generated in this study have been deposited in the Genome Sequence Archive (https://bigd.big.ac.cn/gsa)⁸⁹ in National Genomics Data Center⁹⁰ under accession number CRA012798 [https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA020137]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁹¹ partner repository with the dataset identifier PXD062372. Source data are provided with this paper.

Code availability

Code used for all processing and analysis is available at Github⁹² [https://doi.org/10.5281/zenodo.15100481].

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Acknowledgements

We thank Professor Z.-Z.Gong (CAU) provided pp2c 3 m, Professor J.-G.Huang (SDAU) and Professor J.-K. Zhu (SUSTC) provided snrk2.2/2.3/2.6 seeds, Professor Q.Xie (IGDB, CAS) provides pyr1pyl1/2/4 seeds, Professor H.-C.Cui (NWAFU) provides aba2-1 seeds. We thank Professor W.F. Qian (IGDB, CAS) for supporting ribo-seq analysis, Professor J. S. Zhang (IGDB, CAS) for suggestions of CLIP-seq analysis and Dr. C.Y. Liu (IGDB, CAS) for help with Northern blot assay for pre-rRNA processing. We thank Drs. B.Y. Shi (IoG, CAS) and J. Wang (IGDB, CAS) for suggestions on the experimental conduction and data analysis of Ribo-seq assay. We thank Martin Lewinski (Uni Bielefeld) for providing iCLIP data. We thank the Protein Technology Center of the Chinese Academy of Sciences for their assistance with mass spectrometry. This research is supported by Beijing Natural Science Foundation Outstanding Youth Project (JQ23026), the National Key Research and Development Program of China (2021YFD1201500), the National Natural Science Foundation of China (32170581 and 32370587) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA24010204).

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These authors contributed equally: Jing Zhang, Wenna Shao, Yongxin Xu.

Authors and Affiliations

Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
Jing Zhang, Yongxin Xu, Jinchao Chen, Dongzhi Wang, Xuelei Lin & Jun Xiao
University of Chinese Academy of Sciences, Beijing, China
Jing Zhang, Yongxin Xu, Fa’an Tian, Jinchao Chen & Jun Xiao
School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Wenna Shao & Xiang Yu
Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
Fa’an Tian
Hunan University, Changsha, China
Chongsheng He
John Innes Centre, Norwich Research Park, Norwich, UK
Xiaofei Yang & Yiliang Ding
Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
Xiaofei Yang
RNA Biology and Molecular Physiology, Faculty of Biology, Bielefeld University, Bielefeld, Germany
Dorothee Staiger
CAS-JIC Centre of Excellence for Plant and Microbial Science (CEPAMS), Institute of Genetics and Developmental Biology, CAS, Beijing, China
Jun Xiao

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Contributions

J.X. designed and supervised the research, J.X., J.Z. X.Y. W.-N. S. Y.-X.X. wrote the manuscript. J.Z. did most of the experiments; W.-N.S. Y.-X.X. performed bio-informatics analysis; F.-A.T. constructed pACTIN2::FLAG-eL18y and help with polysome profiling assay; C.-S.H. help with CLIP-seq; D.S.,Y.-L.D., and X.-F.Y. provided pGRP7::GRP7-GFP grp7-1 complementary line and put forward valuable advice on the research; C.-S.H, X.Y., D.-Z.W., X.-L.L., X.-F. Y. D.S. and Y.-L.D. revised the manuscript; J.Z., Y.-X.X., J.-C.C., W.-N. S. X.Y. and J.X. prepared all the figures. All authors discussed the results and commented on the manuscript.

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Correspondence to Xiang Yu or Jun Xiao.

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Nature Communications thanks Shintaro Iwasaki, who co-reviewed with Naohiro Kawamoto, Guoyong Xu and the other, anonymous, reviewers for their contribution to the peer review of this work. A peer review file is available.

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Zhang, J., Shao, W., Xu, Y. et al. Unveiling the regulatory role of GRP7 in ABA signal-mediated mRNA translation efficiency regulation. Nat Commun 16, 3947 (2025). https://doi.org/10.1038/s41467-025-59329-6

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Received: 12 January 2024
Accepted: 15 April 2025
Published: 26 April 2025
DOI: https://doi.org/10.1038/s41467-025-59329-6